Nematode ATP synthase subunit E polypeptide

ABSTRACT

Nucleic acid molecules from nematodes encoding ATP synthase subunit E polypeptides are described. ATP synthase subunit E-like polypeptide sequences are also provided, as are vectors, host cells, and recombinant methods for production of ATP synthase subunit E-like nucleotides and polypeptides. Also described are screening methods for identifying inhibitors and/or activators, as well as methods for antibody production.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.11/091,969, filed Mar. 28, 2005, now abandoned, which is a divisional ofU.S. application Ser. No. 10/160,362, filed May 30, 2002, now U.S. Pat.No. 6,903,190, which claims priority to provisional application Ser. No.60/294,777, filed May 31, 2001, all of which are hereby incorporated byreference.

BACKGROUND

Nematodes (derived from the Greek word for thread) are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved as very successful parasites ofboth plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control., CAB International, NewYork).

Nematode parasites of plants can inhabit all parts of plants, includingroots, developing flower buds, leaves, and stems. Plant parasites areclassified on the basis of their feeding habits into the broadcategories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

The situation is particularly dire for high value crops such asstrawberries and tomatoes where chemicals have been used extensively tocontrol soil pests. The soil fumigant methyl bromide has been usedeffectively to reduce nematode infestations in a variety of thesespecialty crops. It is however regulated under the U.N. MontrealProtocol as an ozone-depleting substance and is scheduled forelimination in 2005 in the US (Carter (2001) California Agriculture,55(3):2). It is expected that strawberry and other commodity cropindustries will be significantly impacted if a suitable replacement formethyl bromide is not found. Presently there are a very small array ofchemicals available to control nematodes and they are frequentlyinadequate, unsuitable, or too costly for some crops or soils (Becker(1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat. No.6,048,714). The few available broad-spectrum nematicides such as Telone(a mixture of 1,3-dichloropropene and chloropicrin) have significantrestrictions on their use because of toxicological concerns (Carter(2001) California Agriculture 55(3):12-18).

Fatty acids are a class of natural compounds that have been investigatedas alternatives to the toxic, non-specific organophosphate, carbamateand fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this general mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including as herbicides (e.g., SCYTHE by Dow Agrosciencesis the C9 saturated fatty acid pelargonic acid), as bactericides andfungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and as insecticides(e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).

The phytotoxicity of fatty acids has been a major constraint on theirgeneral use in agricultural applications (U.S. Pat. No. 5,093,124) andthe mitigation of these undesirable effects while preserving pesticidalactivity is a major area of research. The esterification of fatty acidscan significantly decrease their phytotoxicity (U.S. Pat. Nos.5,674,897; 5,698,592; 6,124,359). Such modifications can however lead todramatic loss of nematicidal activity as is seen for linoleic, linolenicand oleic acid (Stadler et al. (1994) Planta Medica 60(2):128-132) andit may be impossible to completely decouple the phytotoxicity andnematicidal activity of pesticidal fatty acids because of theirnon-specific mode of action. Perhaps not surprisingly, the nematicidalfatty acid pelargonic acid methyl ester (U.S. Pat. Nos. 5,674,897;5,698,592; 6,124,359) shows a relatively small “therapeutic window”between the onset of pesticidal activity and the observation ofsignificant phytotoxicity (Davis et al. (1997) J. Nematol.29(4S):677-684). This is the expected result if both the phytotoxicityand the nematicidial activity derive from the non-specific disruption ofplasma membrane integrity. Similarly the rapid onset of pesticidalactivity seen with many nematicidal fatty acids at therapeuticconcentrations (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) suggestsa non-specific mechanism of action, possibly related to the disruptionof membranes, action potentials and neuronal activity.

Ricinoleic acid, the major component of castor oil, provides anotherexample of the unexpected effects esterification can have on fatty acidactivity. Ricinoleic acid has been shown to have an inhibitory effect onwater and electrolyte absorption using everted hamster jejunal and ilealsegments (Gaginella et al. (1975) J. Pharmacol. Exp. Ther.195(2):355-61) and to be cytotoxic to isolated intestinal epithelialcells (Gaginella et al. (1977) J. Pharmacol. Exp. Ther. 201(1):259-66).These features are likely the source of the laxative properties ofcastor oil which is given as a purgative in humans and livestock. Infact, castor oil is a component of some deworming protocols because ofits laxative properties. In contrast, the methyl ester of ricinoleicacid is ineffective at suppressing water absorption in the hamster model(Gaginella et al. (1975) J. Pharmacol. Exp. Ther. 195(2):355-61).

The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are chemicals that, inprinciple, provide excellent specificity and efficacy and should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two approaches have proven lesseffective for agricultural applications against root pathogens. Althoughcertain avermectins show exquisite activity against plant parasiticnematodes these chemicals are hampered by poor bioavailability due totheir light sensitivity, degradation by soil microorganisms and tightbinding to soil particles (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).Consequently despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

Bt delta toxins must be ingested to affect their target organ the brushborder of midgut epithelial cells (Marroquin et al. (2000) Genetics155(4):1693-1699). Consequently they are not anticipated to be effectiveagainst the dispersal, non-feeding, juvenile stages of plant parasiticnematodes in the field. These juvenile stages only commence feeding whena susceptible host has been infected, thus to be effective nematicidesmay need to penetrate the cuticle. In addition, soil mobility of arelatively large 65-130 kDa protein—the size of typical Bt deltatoxins—is expected to be poor and delivery in planta is likely to beconstrained by the exclusion of large particles by the feeding tube ofcertain plant parasitic nematodes such as Heterodera (Atkinson et al.(1998) Engineering resistance to plant-parasitic nematodes. In: ThePhysiology and Biochemistry of Free-Living and Plant-parasitic Nematodes(eds R. N. Perry & D. J. Wright), CAB International 1998).

Many plant species are known to be highly resistant to nematodes. Themost well documented of these include marigolds (Tagetes spp.),rattlebox (Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.),castor bean (Ricinus communis), margosa (Azardiracta indica), and manymembers of the family Asteraceae (family Compositae) (Hackney &Dickerson (1975) J. Nematol. 7(1):84-90). The active principle(s) forthis nematicidal activity has not been discovered in all of theseexamples and no plant-derived products are sold commercially for controlof nematodes. In the case of the Asteraceae, the photodynamic compoundalpha-terthienyl has been shown to account for the strong nematicidalactivity of the roots. Castor beans are plowed under as a green manurebefore a seed crop is set. However, a significant drawback of the castorplant is that the seed contains toxic compounds (such as ricin) that cankill humans, pets, and livestock and is also highly allergenic.

There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

Nematode parasites of vertebrates (e.g., humans, livestock and companionanimals) include gut roundworms, hookworms, pinworms, whipworms, andfilarial worms. They can be transmitted in a variety of ways, includingby water contamination, skin penetration, biting insects, or byingestion of contaminated food.

In domesticated animals, nematode control or “de-worming” is essentialto the economic viability of livestock producers and is a necessary partof veterinary care of companion animals. Parasitic nematodes causemortality in animals (e.g., heartworm in dogs and cats) and morbidity asa result of the parasites' inhibiting the ability of the infected animalto absorb nutrients. The parasite-induced nutrient deficiency results indiseased livestock and companion animals (i.e., pets), as well as instunted growth. For instance, in cattle and dairy herds, a singleuntreated infection with the brown stomach worm can permanently stunt ananimal's ability to effectively convert feed into muscle mass or milk.

Two factors contribute to the need for novel anthelmintics and vaccinesfor control of parasitic nematodes of animals. First, some of the moreprevalent species of parasitic nematodes of livestock are buildingresistance to the anthelmintic drugs available currently, meaning thatthese products will eventually lose their efficacy. These developmentsare not surprising because few effective anthelmintic drugs areavailable and most have been used continuously. Presently a number ofparasitic species has developed resistance to most of the anthelmintics(Geents et al. (1997) Parasitology Today 13:149-151; Prichard (1994)Veterinary Parasitology 54:259-268). The fact that many of theanthelmintic drugs have similar modes of action complicates matters, asthe loss of sensitivity of the parasite to one drug is often accompaniedby side resistance—that is, resistance to other drugs in the same class(Sangster & Gill (1999) Parasitology Today 15(4):141-146). Secondly,there are some issues with toxicity for the major compounds currentlyavailable.

Human infections by nematodes result in significant mortality andmorbidity, especially in tropical regions of Africa, Asia, and theAmericas. The World Health Organization estimates 2.9 billion people areinfected with parasitic nematodes. While mortality is rare in proportionto total infections (180,000 deaths annually), morbidity is tremendousand rivals tuberculosis and malaria in disability adjusted life yearmeasurements. Examples of human parasitic nematodes include hookworm,filarial worms, and pinworms. Hookworm is the major cause of anemia inmillions of children, resulting in growth retardation and impairedcognitive development. Filarial worm species invade the lymphatics,resulting in permanently swollen and deformed limbs (elephantiasis) andinvade the eyes causing African Riverblindness. Ascaris lumbricoides,the large gut roundworm infects more than one billion people worldwideand causes malnutrition and obstructive bowl disease. In developedcountries, pinworms are common and often transmitted through children indaycare.

Even in asymptomatic parasitic infections, nematodes can still deprivethe host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

While public health measures have nearly eliminated one tropicalnematode (the water-borne Guinea worm), cases of other worm infectionshave actually increased in recent decades. In these cases, drugintervention provided through foreign donations or purchased by thosewho can afford it remains the major means of control. Because of thehigh rates of reinfection after drug therapy, vaccines remain the besthope for worm control in humans. There are currently no vaccinesavailable.

Until safe and effective vaccines are discovered to prevent parasiticnematode infections, anthelmintic drugs will continue to be used tocontrol and treat nematode parasitic infections in both humans anddomestic animals. Finding effective compounds against parasiticnematodes has been complicated by the fact that the parasites have notbeen amenable to culturing in the laboratory. Parasitic nematodes areoften obligate parasites (i.e., they can only survive in theirrespective hosts, such as in plants, animals, and/or humans) with slowgeneration times. Thus, they are difficult to grow under artificialconditions, making genetic and molecular experimentation difficult orimpossible. To circumvent these limitations, scientists have usedCaenorhabidits elegans as a model system for parasitic nematodediscovery efforts.

C. elegans is a small free-living bacteriovorous nematode that for manyyears has served as an important model system for multicellular animals(Burglin (1998) Int. J. Parasitol. 28(3): 395-411). The genome of C.elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282: 2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32:23-30).

Although C. elegans serves as a good model system for vertebrates, it isan even better model for study of parasitic nematodes, as C. elegans andother nematodes share unique biological processes not found invertebrates. For example, unlike vertebrates, nematodes produce and usechitin, have gap junctions comprised of innexin rather than connexin andcontain glutamate-gated chloride channels rather than glycine-gatedchloride channels (Bargmann (1998) Science 282: 2028-33). The latterproperty is of particular relevance given that the avermectin class ofdrugs is thought to act at glutamate-gated chloride receptors and ishighly selective for invertebrates (Martin (1997) Vet. J. 154:11-34).

A subset of the genes involved in nematode specific processes will beconserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of data transfer has been shown in cross phyla comparisons forconserved genes and is expected to be more robust among species within aphylum. Consequently, C. elegans and other free-living nematode speciesare likely excellent surrogates for parasitic nematodes with respect toconserved nematode processes.

Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391:806-811; Montgomery et al. (1998) Proc.Natl. Acad. Sci. USA 95(26):15502-15507). Treatment of a nematode withdouble-stranded RNA of a selected gene can destroy expressed sequencescorresponding to the selected gene thus reducing expression of thecorresponding protein. By preventing the translation of specificproteins, their functional significance and essentiality to the nematodecan be assessed. Determination of essential genes and theircorresponding proteins using C. elegans as a model system will assist inthe rational design of anti-parasitic nematode control products.

SUMMARY

The invention features nucleic acid molecules encoding Meloidogynejavanica, Heterodera glycines, and Zeldia punctata ATP synthase subunitE and other nematode ATP synthase subunit E-like proteins. M. javanicais a Root Knot Nematode that causes substantial damage to several crops,including cotton, tobacco, pepper, and tomato. H. glycines, referred toas Soybean Cyst Nematode, is a major pest of soybean. Z. punctata isfree-living nematode that serves as a model for parasitic nematodes. TheATP synthase subunit E-like nucleic acids and polypeptides of theinvention allow for the identification of a nematode species, and forthe identification of compounds that bind to or alter the activity ofATP synthase subunit E-like polypeptides. Such compounds may provide ameans for combating diseases and infestations caused by nematodes,particularly those caused by M. javanica (e.g., in tobacco, cotton,pepper, or tomato plants) and by H. glycines, (e.g., in soybean).

The invention is based, in part, on the identification of a cDNAencoding M. javanica ATP synthase subunit E (SEQ ID NO: 1). This 466nucleotide cDNA has a 312 nucleotide open reading frame (SEQ ID NO: 7)encoding a 104 amino acid polypeptide (SEQ ID NO: 4).

The invention is also based, in part, on the identification of a cDNAencoding H. glycines ATP synthase subunit E (SEQ ID NO: 2). This 516nucleotide cDNA has a 339 nucleotide open reading frame (SEQ ID NO: 8)encoding a 113 amino acid polypeptide (SEQ ID NO: 5).

The invention is also based, in part, on the identification of a cDNAencoding Z. punctata ATP synthase subunit E (SEQ ID NO: 3). This 489nucleotide cDNA has a 318 nucleotide open reading frame (SEQ ID NO: 9)encoding a 106 amino acid polypeptide (SEQ ID NO: 6).

In one aspect, the invention features novel nematode ATP synthasesubunit E-like polypeptides. Such polypeptides include purifiedpolypeptides having the amino acid sequences set forth in SEQ ID NO: 4,5, and/or 6. Also included are polypeptides having an amino acidsequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, or98% identical to SEQ ID NO: 4, 5, and/or 6. The invention includespolypeptides comprising, consisting of, or consisting essentially ofsuch polypeptides. The invention also features such polypeptides linked,e.g., by a peptide bond to at least one heterologous polypeptide to forma fusion protein. The ATP synthase subunit E-like polypeptide can beflanked by heterologous polypeptides or by one or more heterologousamino acids. The purified polypeptides can be encoded by a nematodegene, e.g., a nematode gene other than C. elegans. For example, thepurified polypeptide has a sequence other than SEQ ID NO: 10 (C. elegansATP synthase subunit E). The purified polypeptides can further include aheterologous amino acid sequence, e.g., an amino-terminal orcarboxy-terminal amino acids (or both) that are not part of thenaturally occurring sequence. Also featured are purified polypeptidefragments of the aforementioned ATP synthase subunit E-likepolypeptides, e.g., a fragment of at least about 20, 30, 40, 50, 75, 85,104, 106, 113 amino acids. Non-limiting examples of such fragmentsinclude: fragments from about amino acid 1 to 50, 1 to 75, 1 to 89, 1 to91, 1 to 99, 1 to 100, 1 to 125, 51 to 113, 93 to 104, 99 to 113, and 93to 106 of SEQ ID NO: 4, 5, and/or 6. The polypeptide or fragment thereofcan be modified, e.g., processed, truncated, modified (e.g. byglycosylation, phosphorylation, acetylation, myristylation, prenylation,palmitoylation, amidation, addition of glycerophosphatidyl inositol), orany combination of the above.

Certain ATP synthase subunit E-like polypeptides comprise a sequence of104, 106, 113, 125, 150 amino acids or fewer.

In another aspect, the invention features novel isolated nucleic acidmolecules encoding nematode ATP synthase subunit E-like polypeptides.Such isolated nucleic acid molecules include nucleic acids having thenucleotide sequence set forth in SEQ ID NO: 1, 2, and/or 3 or SEQ ID NO:7, 8, and/or 9. Also included are isolated nucleic acid molecules havingthe same sequence as or encoding the same polypeptide as a nematode ATPsynthase subunit E-like gene (other than C. elegans ATP synthase subunitE-like genes).

Also featured are: 1) isolated nucleic acid molecules (e.g., nucleicacid probes) having a strand that hybridizes under low stringencyconditions to a single stranded probe of the sequences of SEQ ID NO: 1,2, and/or 3 or their complements and, optionally, encodes polypeptidesof between 104 and 106 or 113 amino acids; 2) isolated nucleic acidmolecules having a strand that hybridizes under high stringencyconditions to a single stranded probe of the sequence of SEQ ID NO: 1,2, and/or 3 or their complements and, optionally, encodes polypeptidesof between 104 and 106 or 113 amino acids; 3) isolated nucleic acidfragments of an ATP synthase subunit E-like nucleic acid molecule, e.g.,a fragment of SEQ ID NO: 1, 2, and/or 3 that is about 280, 415, 420,440, and 500 or more nucleotides in length or ranges between suchlengths; and 4) oligonucleotides that are complementary to an ATPsynthase subunit E-like nucleic acid molecule or an ATP synthase subunitE-like nucleic acid complement, e.g., an oligonucleotide of about 10,15, 18, 20, 22, 24, 28, 30, 35, 40, 50, 60, 70, 80, or more nucleotidesin length. Exemplary oligonucleotides are oligonucleotides which annealto a site located between nucleotides about 1 to 24, 1 to 48, 1 to 60, 1to 120, 24 to 48, 24 to 60, 49 to 60, 61 to 180, 381 to 420, 421 to 480,451 to 466, 451 to 489, and 451 to 516 of SEQ ID NO: 1, 2, and/or 3.Nucleic acid fragments include the following non-limiting examples:nucleotides about 1 to 200, 100 to 300, 200 to 400, 300 to 500, 300 to466, 300 to 516, and 300 to 489 of SEQ ID NO: 1, 2, and/or 3. Alsowithin the invention are nucleic acid molecules that hybridize understringent conditions to nucleic acid molecule comprising SEQ ID NO: 1, 2or 3 and comprise 3,000, 2,000, 1,000 or fewer nucleotides. The isolatednucleic acid can further include a heterologous promoter operably linkedto the ATP synthase subunit E-like nucleic acid molecule.

A molecule featured herein can be from a nematode of the classAraeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida,Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia,Desmoscolecidae, Rhabditida, or Tylenchida. Alternatively, the moleculecan be from a species of the class Rhabditida, particularly a speciesother than C. elegans.

In another aspect, the invention features a vector, e.g., a vectorcontaining an aforementioned nucleic acid. The vector can furtherinclude one or more regulatory elements, e.g., a heterologous promoter.The regulatory elements can be operably linked to the ATP synthasesubunit E-like nucleic acid molecules in order to express an ATPsynthase subunit E-like nucleic acid molecule. In yet another aspect,the invention features a transgenic cell or transgenic organism havingin its genome a transgene containing an aforementioned ATP synthasesubunit E-like nucleic acid molecule and a heterologous nucleic acid,e.g., a heterologous promoter.

In still another aspect, the invention features an antibody, e.g., anantibody, antibody fragment, or derivative thereof that bindsspecifically to an aforementioned polypeptide. Such antibodies can bepolyclonal or monoclonal antibodies. The antibodies can be modified,e.g., humanized, rearranged as a single-chain, or CDR-grafted. Theantibodies may be directed against a fragment, a peptide, or adiscontinuous epitope from an ATP synthase subunit E-like polypeptide.

In another aspect, the invention features a method of screening for acompound that binds to a nematode ATP synthase subunit E-likepolypeptide, e.g., an aforementioned polypeptide. The method includesproviding the nematode polypeptide; contacting a test compound to thepolypeptide; and detecting binding of the test compound to the nematodepolypeptide. In one embodiment, the method further includes contactingthe test compound to a mammalian ATP synthase subunit E-likepolypeptide; and detecting binding of the test compound to the mammalianATP synthase subunit E-like polypeptide. A test compound that binds thenematode ATP synthase subunit E-like polypeptide with at least 2-fold,5-fold, 10-fold, 20-fold, 50-fold, or 100-fold affinity greater relativeto its affinity for the mammalian (e.g., a human) ATP synthase subunitE-like polypeptide can be identified.

The invention also features methods for identifying compounds that alterthe activity of a nematode ATP synthase subunit E-like polypeptide. Themethod includes contacting the test compound to the nematode ATPsynthase subunit E-like polypeptide; and detecting an ATP synthasesubunit E-like activity. A decrease in the level of ATP synthase subunitE-like activity of the polypeptide relative to the level of ATP synthasesubunit E-like activity of the polypeptide in the absence of the testcompound is an indication that the test compound is an inhibitor of theATP synthase subunit E-like activity. In still another embodiment, themethod further includes contacting a test compound such as an allostericinhibitor or other types of inhibitors that prevent binding of the ATPsynthase subunit E-like polypeptide to other molecules or proteins. Achange in activity of proteins normally bound by the subunit E is anindication that the test compound is an inhibitor of the ATP synthasesubunit E-like activity. Such inhibitory compounds are potentialselective agents for reducing the viability of a nematode expressing anATP synthase subunit E-like polypeptide, e.g., the viability of M.javanica, H. glycines, and/or Z. punctata. These methods can alsoinclude contacting the compound with a mammalian (e.g., a human) ATPsynthase subunit E-like polypeptide; and detecting an ATP synthasesubunit E-like activity. A compound that decreases nematode ATP synthasesubunit E activity to a greater extent than it decreases mammalian ATPsynthase subunit E-like polypeptide activity could be useful as aselective inhibitor of the nematode polypeptide. A desirable compoundcan exhibit 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold orgreater selective activity against the nematode polypeptide.

Another featured method is a method of screening for a compound thatalters an activity of an ATP synthase subunit E-like polypeptide oralters binding or regulation of other polypeptides by ATP synthasesubunit E. The method includes providing the polypeptide; contacting atest compound to the polypeptide; and detecting an ATP synthase subunitE-like activity or the activity of polypeptides bound or regulated bythe subunit E (e.g., ATP synthase complex), wherein a change in activityof ATP synthase subunit E-like polypeptides or other downstreampolypeptides relative to the ATP synthase subunit E-like activity of thepolypeptide or downstream polypeptides (e.g., ATP synthase complex) inthe absence of the test compound is an indication that the test compoundalters the activity of the polypeptide(s). The method can furtherinclude contacting the test compound to a mammalian (e.g., a human) ATPsynthase subunit E-like polypeptide and measuring the ATP synthasesubunit E-like activity of the mammalian ATP synthase subunit E-likepolypeptide or other polypeptides affected or regulated by the subunitE. A test compound that alters the activity of the nematode ATP synthasesubunit E-like polypeptide at a given concentration and that does notsubstantially alter the activity of the mammalian ATP synthase subunitE-like polypeptide or downstream polypeptides at the given concentrationcan be identified. An additional method includes screening for bothbinding to an ATP synthase subunit E-like polypeptide and for analteration in the activity of an ATP synthase subunit E-likepolypeptide.

Yet another featured method is a method of screening for a compound thatalters the viability or fitness of a transgenic cell or organism ornematode. The transgenic cell or organism has a transgene that expressesan ATP synthase subunit E-like polypeptide. The method includescontacting a test compound (e.g., an unscreened compound or one known todecrease ATP synthase subunit E activity in vitro) to the transgeniccell or organism and detecting changes in the viability or fitness ofthe transgenic cell or organism. This alteration in viability or fitnesscan be measured relative to an otherwise identical cell or organism thatdoes not harbor the transgene.

Also featured is a method of screening for a compound that alters theexpression of a nematode nucleic acid encoding an ATP synthase subunitE-like polypeptide, e.g., a nucleic acid encoding a M. javanica, H.glycines, and/or Z. punctata ATP synthase subunit E-like polypeptide.The method includes contacting a cell, e.g., a nematode cell, with atest compound and detecting expression of a nematode nucleic acidencoding an ATP synthase subunit E-like polypeptide, e.g., byhybridization to a probe complementary to the nematode nucleic acidencoding an ATP synthase subunit E-like polypeptide or by contactingpolypeptides isolated from the cell with a compound, e.g., antibody thatbinds an ATP synthase subunit E-like polypeptide. Compounds identifiedby the method are also within the scope of the invention.

The screening methods described herein can further include exposing anematode to the compound and assessing the effect of the compound on theviability or reproductive ability of the nematode. Such methods canentail exposing nematodes to those compounds which bind to, inhibit,reduce the expression of or otherwise interfere with ATP synthasesubunit E-like activity. Compounds which reduce nematode viability orreproductive ability in such assays are candidate pesticides.

In yet another aspect, the invention features a method of treating adisorder (e.g., an infection) caused by a nematode, e.g., M. javanica orH. glycines, in a subject, e.g., a host plant or host animal. The methodincludes administering to the subject an effective amount of aninhibitor of an ATP synthase subunit E-like polypeptide activity or aninhibitor of expression of an ATP synthase subunit E-like polypeptide.Non-limiting examples of such inhibitors include: an antisense nucleicacid (or PNA) to an ATP synthase subunit E-like nucleic acid, anantibody to an ATP synthase subunit E-like polypeptide, or a smallmolecule identified as an ATP synthase subunit E-like polypeptideinhibitor by a method described herein.

A “purified polypeptide”, as used herein, refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it is naturally associated. The polypeptide can constitute atleast 10, 20, 50 70, 80 or 95% by dry weight of the purifiedpreparation.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three, preferably one, separate genes. The termtherefore covers, for example: (a) a DNA which is part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the nucleicacid sequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in mixtures of different (i) DNA molecules, (ii)transfected cells, or (iii) cell clones in a DNA library such as a cDNAor genomic DNA library. Isolated nucleic acid molecules according to thepresent invention further include molecules produced synthetically, aswell as any nucleic acids that have been altered chemically and/or thathave modified backbones.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”refers to the sequence of the nucleotides in the nucleic acid molecule,the two phrases can be used interchangeably.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the BLASTN and BLASTX programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the BLASTX program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Wheregaps exist between two sequences, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., BLASTX and BLASTN) can be used.Available on the internet at ncbi.nlm.nih.gov.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one or more subject polypeptides), which is partly orentirely heterologous, i.e., foreign, to the transgenic plant, animal,or cell into which it is introduced, or, is homologous to an endogenousgene of the transgenic plant, animal, or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe plant's genome in such a way as to alter the genome of the cell intowhich it is inserted (e.g., it is inserted at a location which differsfrom that of the natural gene or its insertion results in a knockout). Atransgene can include one or more transcriptional regulatory sequencesand other nucleic acid sequences, such as introns, that may be necessaryfor optimal expression of the selected nucleic acid, all operably linkedto the selected nucleic acid, and may include an enhancer sequence.

As used herein, the term “transgenic cell” refers to a cell containing atransgene.

As used herein, a “transgenic plant” is any plant in which one or more,or all, of the cells of the plant includes a transgene. The transgenecan be introduced into the cell, directly or indirectly by introductioninto a precursor of the cell, by way of deliberate genetic manipulation,such as by T-DNA mediated transfer, electroporation, or protoplasttransformation. The transgene may be integrated within a chromosome, orit may be extrachromosomally replicating DNA.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which affects expressionof the selected DNA sequence in specific cells of a tissue, such as aleaf, root, or stem.

As used herein, the terms “hybridizes under stringent conditions” and“hybridizes under high stringency conditions” refer to conditions forhybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60° C. or65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6×SSC buffer atabout 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at50° C.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

As used herein, an agent with “anthelminthic activity” is an agent,which when tested, has measurable nematode-killing activity or resultsin infertility or sterility in the nematodes such that unviable or nooffspring result. In the assay, the agent is combined with nematodes,e.g., in a well of microtiter dish having agar media or in the soilcontaining the agent. Staged adult nematodes are placed on the media.The time of survival, viability of offspring, and/or the movement of thenematodes are measured. An agent with “anthelminthic activity” reducesthe survival time of adult nematodes relative to unexposed similarlystaged adults, e.g., by about 20%, 40%, 60%, 80%, or more. In thealternative, an agent with “anthelminthic activity” may also cause thenematodes to cease replicating, regenerating, and/or producing viableprogeny, e.g., by about 20%, 40%, 60%, 80%, or more.

As used herein, the term “binding” refers to the ability of a firstcompound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.

As used herein, the term “binds specifically” refers to the ability ofan antibody to discriminate between a target ligand and a non-targetligand such that the antibody binds to the target ligand and not to thenon-target ligand when simultaneously exposed to both the given ligandand non-target ligand, and when the target ligand and the non-targetligand are both present in molar excess over the antibody.

As used herein, the term “altering an activity” refers to a change inlevel, either an increase or a decrease in the activity, (e.g., anincrease or decrease in the ability of the polypeptide to bind orregulate other polypeptides or molecules) particularly an ATP synthasesubunit E-like or ATP synthase subunit E activity. The change can bedetected in a qualitative or quantitative observation. If a quantitativeobservation is made, and if a comprehensive analysis is performed over aplurality of observations, one skilled in the art can apply routinestatistical analysis to identify modulations where a level is changedand where the statistical parameter, the p value, is less than 0.05.

In part, the nematode ATP synthase subunit E proteins and nucleic acidsdescribed herein are novel targets for anti-nematode vaccines,pesticides, and drugs. Inhibition of these molecules can provide meansof inhibiting nematode metabolism and/or the nematode life-cycle.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the cDNA sequence of M. javanica ATP synthase subunit E(SEQ ID NO: 1), its corresponding encoded amino acid sequence (SEQ IDNO: 4), and its open reading frame (SEQ ID NO: 7).

FIG. 2 depicts the cDNA sequence of H. glycines ATP synthase subunit E(SEQ ID NO: 2), its corresponding encoded amino acid sequence (SEQ IDNO: 5), and its open reading frame (SEQ ID NO: 8).

FIG. 3 depicts the cDNA sequence of Z. punctata ATP synthase subunit E(SEQ ID NO: 3), its corresponding encoded amino acid sequence (SEQ IDNO: 6), and its open reading frame (SEQ ID NO: 9).

FIG. 4 is an alignment of the sequences of M. javanica, H. glycines, andZ. punctata ATP synthase subunit E-like polypeptides (SEQ ID NO: 4, 5,and 6) and C. elegans ATP synthase subunit E-like polypeptide (SEQ IDNO: 10).

DETAILED DESCRIPTION

ATP synthases of eubacteria, chloroplasts, and mitochondria synthesizeATP from ADP and inorganic phosphate using a transmembrane protongradient to drive the reaction. In bacterial enzymes and inreconstituted mitochondrial enzymes the process is reversible and theenzymes can also hydrolyze ATP and use the energy released in theprocess to pump protons. The enzymes from various sources differ incomplexity of their subunits. To date, the simplest ATP synthase to bedescribed (F₁F₀ synthase) is from E. coli. The F₁F₀ synthase has eightdifferent subunits. Five of the subunits form a globular catalyticsubcomplex (F₁), and three others comprise the membrane bound domain ofthe enzyme (F₀) to which the catalytic F₁ subcomplex is bound. Protonflux through the F₀ subcomplex has been postulated to causeconformational changes, which may pass to the catalytic F₁ subcomplexthrough the stalk of the F₀ complex. While the overall architecture ofthe ATP synthases in higher invertebrates and vertebrates appears to besimilar to that of bacterial ATP synthases, they are generally morecomplex and have a number of additional subunits. Mammalianmitochondrial ATP synthases, for example, include between 12 and 18protein components (Walter et al. (1991) Biochemistry 30: 5369-5378).

One subunit suspected of having a regulatory role in a mammalian ATPsynthases, perhaps in response to Ca²⁺, is subunit E. Subunit E is ahighly charged, basic protein that has been shown to be peripherallyassociated with the F₀ subcomplex of the mammalian F₁F₀-ATP synthase.Subunit E is thought to bind to the F₀ subcomplex and transmitconformational changes to the F₁ catalytic subcomplex. The regulatoryrole of subunit E is predicted based upon its differential regulation atthe transcriptional level in response to such diverse conditions ashypoxia, UV irradiation, and high/low fat diets. Ultimately, regulationof the F₀F₁-ATP synthase, through subunit E and other subunits, leads tocontrol of energy production, as would be expected of an enzyme involvedin ATP synthesis (Elliot et al. (1993) Biochem Biophys. Res. Com.190:167-174; Levy (1997) Amer. Phys. Soc. 457-465).

This invention describes a novel class of nematode genes related C.elegans protein T23910 (GenBank® Accession No: 7506279). The nematodegenes can be shown by a PSI-BLAST bioinformatics analysis to be highlydivergent members of the ATP synthase subunit E gene family. Thisdivergent gene family appears to be restricted to higher metazoans(e.g., nematodes, arthropods, vertebrates) and is not detected inavailable sequences of fungi, bacteria or plants. We have identifiedadditional homologs in the nematodes M. javanica, H. glycines and Z.punctata. Importantly, we have shown that these proteins are essentialfor the viability of C. elegans using RNAi interference, suggesting thatthese proteins are promising targets for anti-parasitic compounds.

As in the case of the mammalian proteins, the nematode homologs aresmall, hydrophilic proteins. Despite the low pairwise sequence identityover the entire length of molecule (below 30%) for severalnematode-vertebrate comparisons, a multiple alignment of all ATPsynthase subunit E-like proteins shows regions of similarity, as well asabsolute conservation in some regions (particularly in the aminoterminus). Another quality shared among the members of this family isthe lack of a mitochondrial pro-sequence. Instead, the proteins are allpredicted to contain putative transmembrane regions in their N-terminalregions (by TMHMM, available on the Internet atcbs.dtu.dk/services/TMHMM/), which can be recognized as a weakpreference for mitochondrial localization in some cases (by Target P,available on the Internet at cbs.dtu.dk/services/TargetP/).

The present invention provides nucleic acids from nematodes encoding ATPsynthase subunit E-like polypeptides. The M. javanica nucleic acidmolecule (SEQ ID NO: 1) and the encoded ATP synthase subunit E-likepolypeptide (SEQ ID NO: 4) are depicted in FIG. 1. The H. glycinesnucleic acid molecule (SEQ ID NO: 2) and the ATP synthase subunit E-likepolypeptide (SEQ ID NO: 5) are depicted in FIG. 2. The Z. punctatanucleic acid molecule (SEQ ID NO: 3) and the ATP synthase subunit E-likepolypeptide (SEQ ID NO: 6) are depicted in FIG. 3. Certain sequenceinformation for the ATP synthase subunit E genes described herein issummarized in Table 1, below.

TABLE 1 ATP Synthase Subunit E Sequences Species cDNA ORF PolypeptideFIG. M. javanica SEQ ID NO: 1 SEQ ID NO: 7 SEQ ID NO: 4 FIG. 1 H.glycines SEQ ID NO: 2 SEQ ID NO: 8 SEQ ID NO: 5 FIG. 2 Z. punctata SEQID NO: 3 SEQ ID NO: 9 SEQ ID NO: 6 FIG. 3

The invention is based, in part, on the discovery of ATP synthasesubunit E-like sequences from M. javanica, H. glycines, and Z. punctata.The following examples are, therefore, to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. All of the publications cited herein are herebyincorporated by reference in their entirety.

EXAMPLES

A TBLASTN query with the C. elegans gene T23910 (GenBank® GI: 7506279)identified multiple expressed sequence tags (ESTs are short nucleic acidfragment sequences from single sequencing reads) in dbest that arepredicted to encode a portion of ATP synthase subunit E-like enzymes inat least three nematode species: M. javanica (GenBank® GI: 9829776)similar to C. elegans codons 12-104; H. glycines (GenBank® GI: 10713753)similar to C. elegans codons 6-104; and Z. punctata (GenBank® GI:7710479) similar to C. elegans codons 15-107, all from McCarter, et al.(1999) Washington University Nematode EST Project. Also identified weresequences from Pristionchus pacificus (GenBank® Identification No:5914683) similar to C. elegans codons 6-107; Strongyloides stercoralis(GenBank® GI: 10715244) similar to C. elegans codons 1-107; Ancylostomacaninum (GenBank® GI: 11180617) similar to C. elegans codons 49-107 (allfrom McCarter et al. (1999) Washington University Nematode EST Project),Litomosoides sigmodontis (GenBank® GI: 6200636) similar to C. eleganscodons 1-58 (from Allen et al. (2000) Infect. Immun. 68:5454-8); andBrugia malayi (GenBank® GI: 1592572) similar to C. elegans codons 8-58were also identified in dbest.

Full Length ATP Synthase Subunit E-Like cDNA Sequences

Plasmid clone Div348, corresponding to the M. javanica EST sequence (GI:9829776) was obtained from the Genome Sequencing Center (St. Louis,Mo.). Similarly, plasmid clone Div361, corresponding to the H. glycinesEST sequence (GI: 10713753), and plasmid clone Div222, corresponding tothe Z. punctata EST sequence (GI: 7710479), were also obtained from theGenome Sequencing Center (St. Louis, Mo.). The cDNA inserts in theplasmids were sequenced in their entirety to obtain full-lengthsequences for ATP synthase subunit E-like genes from M. javanica (SEQ IDNO: 1), H. glycines (SEQ ID NO:2), and Z. punctata (SEQ ID NO:3).

Unless otherwise indicated, all nucleotide sequences determined hereinwere sequenced with an automated DNA sequencer (such as model 373 fromApplied Biosystems, Inc.) using processes well-known to those skilled inthe art. Primers used for sequencing are listed in Table 2, below.

TABLE 2 Sequencing Primers SEQ ID Name Sequence NO: Homology to T7gtaatacgactcactatagggc 11 vector polylinker primer T3aattaaccctcactaaaggg 12 vector polylinker primer SL1gggtttaattacccaagtttga 13 nematode transpliced leader Oligo dTgagagagagagagagagagaactagtctcgagtttttttttttttttttt 14 universal primerto poly A tailCharacterization of M. javanica, H. glycines, and Z. punctata ATPSynthase Subunit E

The sequences of three ATP synthase subunit E-like nucleic acidmolecules are depicted in FIG. 1, FIG. 2, and FIG. 3 as SEQ ID NO: 1,SEQ ID NO: 2, and SEQ ID NO: 3, respectively. SEQ ID NO: 7 contains anopen reading frame encoding a 104 amino acid polypeptide, SEQ ID NO: 8contains open reading frame encoding a 113 amino acid polypeptide, andSEQ ID NO: 9 contains an open reading frame encoding a 106 amino acidpolypeptide.

The sequence of the M. javanica ATP synthase subunit E-like cDNA (SEQ IDNO: 1) is depicted in FIG. 1. This nucleotide sequence also contains anopen reading frame (SEQ ID NO:7) encoding a 104 amino acid polypeptide(SEQ ID NO:4). The M. javanica ATP synthase subunit E-like proteinsequence (SEQ ID NO: 4) is also approximately 38% identical to the C.elegans ATP synthase subunit E-like gene (SEQ ID NO: 10).

The sequence of the H. glycines ATP synthase subunit E-like cDNA (SEQ IDNO:2) is depicted in FIG. 2. This nucleotide sequence contains an openreading frame (SEQ ID NO:8) encoding a 113 amino acid polypeptide (SEQID NO:5). The H. glycines ATP synthase subunit E-like protein sequence(SEQ ID NO: 5) is approximately 41% identical to the C. elegans ATPsynthase subunit E-like gene (SEQ ID NO: 10).

The sequence of the Z. punctata ATP synthase subunit E-like cDNA (SEQ IDNO:3) is depicted in FIG. 3. This nucleotide sequence contains an openreading frame (SEQ ID NO:9) encoding a 106 amino acid polypeptide (SEQID NO:6). The Z. punctata ATP synthase subunit E-like protein sequence(SEQ ID NO: 6) is approximately 36% identical to the C. elegans ATPsynthase subunit E-like gene (SEQ ID NO: 10).

The similarity among the M. javanica, H. glycines, Z. punctata, and C.elegans polypeptides is presented as a multiple alignment generated byClustal X multiple alignment program as described below (FIG. 4).

The similarity between M. javanica, H. glycines, and Z. punctata ATPsynthase subunit E-like sequences and other sequences was alsoinvestigated by comparison to sequence databases using BLASTP analysisagainst nr (a non-redundant protein sequence database available on theInternet at ncbi.nlm.nih.gov/) and TBLASTN analysis against dbest (anEST sequence database available on the Internet at ncbi.nlm.nih.gov/;top 500 hits; E=1e-4). The “Expect (E) value” is the number of sequencesthat are predicted to align by chance to the query sequence with a scoreS or greater given the size of the database queried. This analysis wasused to determine the potential number of plant and vertebrate homologsfor each of the nematode ATP synthase subunit E-like polypeptidesdescribed above. M. javanica (SEQ ID NO: 1), H. glycines (SEQ ID NO: 2),Z. punctata (SEQ ID NO: 3), and C. elegans (SEQ ID NO: 10) ATP synthasesubunit E-like sequences had no vertebrate and/or plant hits in nr ordbest having sufficient sequence similarity to meet the threshold Evalue of 1e-4 (this E value approximately corresponds to a threshold forremoving sequences having a sequence identity of less than about 25%over approximately 100 amino acids). Accordingly, the M. javanica, H.glycines, and/or Z. punctata ATP synthase subunit E-like enzymes of thisinvention do not appear to share significant sequence similarity withthe more common vertebrate forms of the enzyme such as the Homo sapiens(GenBank® GI: 6005717; GenBank® Accession No: NP_(—)009031.1) or theRattus norvegicus (GenBank® Accession No: P29419) ATP synthase subunitE.

On the basis of the lack of similarity to plants and vertebrates, the M.javanica, H. glycines, and/or Z. punctata ATP synthase subunit E-likeenzymes are useful targets of inhibitory compounds selective for somenematodes over their hosts (e.g., humans, animals, and plants).

Functional predictions were made using four iterations of PSI-BLAST withthe default parameters on the nr database. PSI-BLAST searches andmultiple alignment construction with CLUSTALX demonstrated that the Celegans gene (GenBank® Accession No: T23910) was a member of the ATPsynthase subunit E family. Reciprocal blast searches and phylogenetictrees confirm that the nucleotide sequences in M. javanica, H. glycines,and/or Z. punctata do encode orthologs of the C. elegans gene andtherefore also likely ATP synthase subunit E proteins. Proteinlocalizations were predicted using the TargetP server available on theInternet at cbs.dtu.dk/services/TargetP/). The M. javanica, H. glycines,and/or Z. punctata ATP synthase subunit E (SEQ ID NO: 4, 5, and 6,respectively) polypeptides are potentially mitochondrial based on thepresence of putative transmembrane domain in the amino-terminus and thefact that all other proteins in the family have weak mitochondrialsignals and putative transmembrane domains in the N-terminus.

RNA Mediated Interference (RNAi) A double stranded RNA (dsRNA) moleculecan be used to inactivate a subunit E-like gene in a cell by a processknown as RNA mediated-interference (Fire et al. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a subunit E-like nucleicacid described herein or a fragment thereof. For example, the moleculecan comprise at least 50, at least 100, at least 200, at least 300, orat least 500 or more contiguous nucleotides of a subunit E-like gene.The dsRNA molecule can be delivered to nematodes via direct injection,by soaking nematodes in aqueous solution containing concentrated dsRNA,or by raising bacteriovorous nematodes on E. coli genetically engineeredto produce the dsRNA molecule (Kamath et al. (2000) Genome Biol. 2;Tabara et al. (1998) Science 282:430-431).

C. elegans were grown on lawns of E. coli genetically engineered toproduce double stranded RNA designed to inhibit ATP synthase subunitexpression. E. coli were transformed with a 437 nucleotide genomicfragment of the subunit E-like gene. The genomic fragment included 255nucleotides of exon sequence and 182 nucleotides of intron sequence (58%exon overall). The exonic sequences correspond to the first 115nucleotides of SEQ ID NO:4, followed by 182 nucleotides of intronicsequence (interrupting the glycine codon at position 39) and then by 140nucleotides of additional exonic sequence (ending at the glutamine codonat position 85). The 437 nucleotide genomic fragment was cloned into anE. coli expression vector between opposing T7 polymerase promoters, andthe vector was transformed into a strain of E. coli that carries anIPTG-inducible T7 polymerase. As a control, E. coli was transformed witha gene encoding the Green Fluorescent Protein (GFP). GFP is a commonlyused reporter gene originally isolated from jellyfish and is widely usedin both prokaryotic and eukaryotic systems. The GFP gene is not presentin the wild-type C. elegans genome and thus it does not trigger an RNAiphenotype when ingested by C. elegans. In both samples, C. elegans wasgrown at 15° C. on NGM plates containing IPTG and E. coli expressing thesubunit E-like specific dsRNA or GFP. Total eggs layed and hatch-ratesof F1 and F2 individuals were followed over the course of 7-10 days (asshown below) and compared to nematode cultures grown on non-toxicdsRNAs.

In another example, dsRNA was injected into the nematode, basically asdescribed in Mello et al. (1991) EMBO J. 10:3959-3970. In short, aplasmid was constructed that contains a portion of the C. elegans genesequence, specifically a fragment 437 nucleotides long, containing 115nucleotides of the first exon followed by the first intron of 182nucleotides and 140 nucleotides of the second exon (58% exon sequence)corresponding to amino acid positions 1-85. The TOPO vector and PCRprimers corresponding to the T7 and SP6 regions were to specificallyamplify this sequence as a linear dsDNA. Single-strand RNAs can betranscribed from this fragment using either T7 RNA polymerase or SP6 RNApolymerase (the RNAs correspond to the sense and antisense RNA strands).RNA so produced was precipitated and resuspended in RNAse free water.SsRNAs were combined, heated to 95° C. for two minutes then allowed tocool from 70° C. to room temperature over 1.5-2.5 hours.

DsRNA was injected into the body cavity of 15-20 young adult C. eleganshermaphrodites. Worms were typically immobilized on an agarose pad andinjected with 2-5 nanoliters of dsRNA at a concentration of 1 mg/ml.Injections were performed with visual observation using a Zeiss Axiovertcompound microscope equipped with 10× and 40×DIC objectives. Needles formicroinjection were prepared using a Narishige needle puller, stagemicromanipulator (Leitz) and an N2-powered injector (Narishige) set at10-20 p.s.i. After injection, 200 μl of recovery buffer (0.1% salmonsperm DNA, 4% glucose, 2.4 mM KCl, 66 mM NaCl, 3 mM CaCl₂, 3 mM HEPES,pH 7.2) was added to the agarose pad and the worms were allowed torecover on the agarose pad for 0.5-4 hours. After recovery, the wormswere transferred to NGM agar plates seeded with a lawn of E. coli strainOP50 as a food source. The following day and for 3 successive daysthereafter, 7 individual healthy injected worms were transferred to newNGM plates seeded with OP50. The number of eggs laid per worm per dayand the number of those eggs that hatch and reach fertile adulthood canbe determined. As a control, GFP dsRNA was produced and injected usingsimilar methods.

The results of the studies described above were as follows.

Feeding RNAi:

Experiment F8-D403 (ATP synthetase subunit E-like RNA)

-   Total # worms monitored: 6-   Total # eggs layed: 171-   Total # eggs hatched: 2-   Hatch %: 1.2%    Experiment F8-D334 (GFP control RNA)-   Total # worms monitored: 6-   Total # eggs layed: 527-   Total # eggs hatched: 526-   Hatch %: 99.8%    Injection RNAi:    Experiment J332 (ATP synthetase subunit-like RNA)-   Total # worms monitored: 7-   Total # eggs layed: 141-   Total # eggs hatched: 0-   Hatch %: 0.0%    Experiment J335 (GFP control RNA)-   Total # worms monitored: 8-   Total # eggs layed: 798-   Total # eggs hatched: 789-   Hatch %: 98.9%

As the results demonstrate, C. elegans cultures grown in the presence ofE. coli expressing dsRNA and those injected with dsRNA from the subunitE-like gene were strongly impaired indicating that the subunit E-likegene provides an essential function in nematodes and that dsRNA from thesubunit E-like gene is lethal when ingested by or injected into C.elegans.

These results demonstrate that ATP synthase subunit E is important forthe viability of C. elegans and suggest that it is a useful target forthe development of compounds that reduce the viability of nematodes.

Identification of Additional ATP Synthase Subunit E-Like Sequences

A skilled artisan can utilize the methods provided in the example aboveto identify additional nematode ATP synthase subunit E-like sequences,e.g., ATP synthase subunit E-like sequence from nematodes other than M.javanica, H. glycines, Z. punctata and/or C. elegans. In addition,nematode ATP synthase subunit E-like sequences can be identified by avariety of methods including computer-based database searches,hybridization-based methods, and functional complementation.

Database Identification. A nematode ATP synthase subunit E-like sequencecan be identified from a sequence database, e.g., a protein or nucleicacid database using a sequence disclosed herein as a query. Sequencecomparison programs can be used to compare and analyze the nucleotide oramino acid sequences. One such software package is the BLAST suite ofprograms from the National Center for Biotechnology Institute (NCBI;Altschul et al. (1997) Nucl. Acids Research 25:3389-3402). An ATPsynthase subunit E-like sequence of the invention can be used to query asequence database, such as nr, dbest (expressed sequence tag (EST)sequences), and htgs (high-throughput genome sequences), using acomputer-based search, e.g., FASTA, BLAST, or PSI-BLAST search.Homologous sequences in other species (e.g., humans and animals) can bedetected in a PSI-BLAST search of a database such as nr (E value=10, Hvalue=1e-2, using, for example, four iterations; available on theInternet at ncbi.nlm.nih.gov/). Sequences so obtained can be used toconstruct a multiple alignment, e.g., a ClustalX alignment, and/or tobuild a phylogenetic tree, e.g., in ClustalX using the Neighbor-Joiningmethod (Saitou et al. (1987) Mol. Biol. Evol. 4:406-425) andbootstrapping (1000 replicates; Felsenstein (1985) Evolution39:783-791). Distances may be corrected for the occurrence of multiplesubstitutions [D_(corr)=−ln(1−D−D²/5) where D is the fraction of aminoacid differences between two sequences] (Kimura (1983) The NeutralTheory of Molecular Evolution, Cambridge University Press).

The aforementioned search strategy can be used to identify ATP synthasesubunit E-like sequences in nematodes of the following non-limiting,exemplary genera: Plant nematode genera: Afrina, Anguina,Aphelenchoides, Belonolaimus, Bursaphelenchus, Cacopaurus, Cactodera,Criconema, Criconemoides, Cryphodera, Ditylenchus, Dolichodorus,Dorylaimus, Globodera, Helicotylenchus, Hemicriconemoides,Hemicycliophora, Heterodera, Hirschmanniella, Hoplolaimus, Hypsoperine,Longidorus, Meloidogyne, Mesoanguina, Nacobbus, Nacobbodera,Panagrellus, Paratrichodorus, Paratylenchus, Pratylenchus,Pterotylenchus, Punctodera, Radopholus, Rhadinaphelenchus,Rotylenchulus, Rotylenchus, Scutellonema, Subanguina, Thecavermiculatus,Trichodorus, Turbatrix, Tylenchorhynchus, Tylenchulus, Xiphinema.

Animal and human nematode genera: Acanthocheilonema, Aelurostrongylus,Ancylostoma, Angiostrongylus, Anisakis, Ascaris, Ascarops, Bunostomum,Brugia, Capillaria, Chabertia, Cooperia, Crenosoma, Cyathostome species(Small Strongyles), Dictyocaulus, Dioctophyma, Dipetalonema,Dirofiliaria, Dracunculus, Draschia, Elaneophora, Enterobius,Filaroides, Gnathostoma, Gonylonema, Habronema, Haemonchus,Hyostrongylus, Lagochilascaris, Litomosoides, Loa, Mammomonogamus,Mansonella, Muellerius, Metastrongylid, Necator, Nematodirus,Nippostrongylus, Oesophagostomum, Ollulanus, Onchocerca, Ostertagia,Oxyspirura, Oxyuris, Parafilaria, Parascaris, Parastrongyloides,Parelaphostrongylus, Physaloptera, Physocephalus, Protostrongylus,Pseudoterranova, Setaria, Spirocerca, Stephanurus, Stephanofilaria,Strongyloides, Strongylus, Spirocerca, Syngamus, Teladorsagia, Thelazia,Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris,Uncinaria, and Wuchereria.

Particularly preferred nematode genera include: Plant: Anguina,Aphelenchoides, Belonolaimus, Bursaphelenchus, Ditylenchus,Dolichodorus, Globodera, Heterodera, Hoplolaimus, Longidorus,Meloidogyne, Nacobbus, Pratylenchus, Radopholus, Rotylenchus,Tylenchulus, Xiphinema.

Animal and human: Ancylostoma, Ascaris, Brugia, Capillaria, Cooperia,Cyathostome species, Dictyocaulus, Dirofiliaria, Dracunculus,Enterobius, Haemonchus, Necator, Nematodirus, Oesophagostomum,Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parascaris, Strongyloides,Strongylus, Syngamus, Teladorsagia, Thelazia, Toxocara, Trichinella,Trichostrongylus, Trichuris, and Wuchereria.

Particularly preferred nematode species include: Plant: Anguina tritici,Aphelenchoides fragariae, Belonolaimus longicaudatus, Bursaphelenchusxylophilus, Ditylenchus destructor, Ditylenchus dipsaci Dolichodorusheterocephalous, Globodera pallida, Globodera rostochiensis, Globoderatabacum, Heterodera avenae, Heterodera cardiolata, Heterodera carotae,Heterodera cruciferae, Heterodera glycines, Heterodera major, Heteroderaschachtii, Heterodera zeae, Hoplolaimus tylenchiformis, Longidorussylphus, Meloidogyne acronea, Meloidogyne arenaria, Meloidogynechitwoodi, Meloidogyne exigua, Meloidogyne graminicola, Meloidogynehapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne nassi,Nacobbus batatiformis, Pratylenchus brachyurus, Pratylenchus coffeae,Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae,Radopholus similis, Rotylenchus reniformis, Tylenchulus semipenetrans,Xiphinema americanum.

Animal and human: Ancylostoma braziliense, Ancylostoma caninum,Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma tubaeforme,Ascaris suum, Ascaris lumbrichoides, Brugia malayi, Capillaria bovis,Capillaria plica, Capillaria feliscati, Cooperia oncophora, Cooperiapunctata, Cyathostome species, Dictyocaulus filaria, Dictyocaulusviviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis, Dracunculusinsignis, Enterobius vermicularis, Haemonchus contortus, Haemonchusplacei, Necator americanus, Nematodirus helvetianus, Oesophagostomumradiatum, Onchocerca volvulus, Onchocerca cervicalis, Ostertagiaostertagi, Ostertagia circumcincta, Oxyuris equi, Parascaris equorum,Strongyloides stercoralis, Strongylus vulgaris, Strongylus edentatus,Syngamus trachea, Teladorsagia circumcincta, Toxocara cati, Trichinellaspiralis, Trichostrongylus axei, Trichostrongylus colubriformis,Trichuris vulpis, Trichuris suis, Trichurs trichiura, and Wuchereriabancrofti.

Further, an ATP synthase subunit E-like sequence can be used to identifyadditional ATP synthase subunit E-like sequence homologs within agenome. Multiple homologous copies of an ATP synthase subunit E-likesequence can be present. For example, a nematode ATP synthase subunitE-like sequence can be used as a seed sequence in an iterative PSI-BLASTsearch (default parameters, substitution matrix=Blosum62, gap open=11,gap extend=1) of a non redundant database such as wormpep (E value=1e-2,H value=1e-4, using, for example 4 iterations) to determine the numberof homologs in a database, e.g., in a database containing the completegenome of an organism. A nematode ATP synthase subunit E-like sequencecan be present in a genome along with 1, 2, 3, 4, 5, 6, 8, 10, or morehomologs.

Hybridization Methods. A nematode ATP synthase subunit E-like sequencecan be identified by a hybridization-based method using a sequenceprovided herein as a probe. For example, a library of nematode genomicor cDNA clones can be hybridized under low stringency conditions withthe probe nucleic acid. Stringency conditions can be modulated to reducebackground signal and increase signal from potential positives. Clonesso identified can be sequenced to verify that they encode ATP synthasesubunit E-like sequences.

Another hybridization-based method utilizes an amplification reaction(e.g., the polymerase chain reaction (PCR)). Oligonucleotides, e.g.,degenerate oligonucleotides, are designed to hybridize to a conservedregion of an ATP synthase subunit E-like sequence (e.g., a regionconserved in the three nematode sequences depicted in FIG. 4). Theoligonucleotides are used as primers to amplify an ATP synthase subunitE-like sequence from template nucleic acid from a nematode, e.g., anematode other than M. javanica, H. glycines, Z. punctata, and/or C.elegans. The amplified fragment can be cloned and/or sequenced.

Complementation Methods. A nematode ATP synthase subunit E-like sequencecan be identified from a complementation screen for a nucleic acidmolecule that restores ATP synthase subunit E-like activity to a celllacking an ATP synthase subunit E-like activity. Routine methods can beused to construct strains (i.e., nematode strains) that lack specificenzymatic activities, e.g., ATP synthase subunit E activity. Forexample, a nematode strain mutated at the ATP synthase subunit E genelocus can be identified by selecting for resistance to inhibitorycompounds and/or compounds that prevent the subunit E from binding toand thus, regulating, activity of an ATP synthase. Such a strain can betransformed with a plasmid library expressing nematode cDNAs. Strainscan be identified in which ATP synthase subunit E activity is restored.For example, the ATP synthase subunit E mutant strains transformed withthe plasmid library can be exposed to allosteric inhibitors or otherinhibitory compounds to select for strains that have acquiredsensitivity to the inhibitors and are expressing a nematode ATP synthasesubunit E-like gene. The plasmid harbored by the strain can be recoveredto identify and/or characterize the inserted nematode cDNA that providesATP synthase subunit E-like activity when expressed.

Full-length cDNA and Sequencing Methods. The following methods can beused, e.g., alone or in combination with another method describedherein, to obtain full-length nematode ATP synthase subunit E-like genesand determine their sequences.

Plant parasitic nematodes are maintained on greenhouse pot culturesdepending on nematode preference. Root Knot Nematodes (Meloidogyne sp)are propagated on Rutgers tomato (Burpee), while Soybean Cyst Nematodes(Heterodera sp) are propagated on soybean. Total nematode RNA isisolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packedworms are combined with 8 ml TRIZOL reagent and solubilized byvortexing. Following 5 minutes of incubation at room temperature, thesamples are divided into smaller volumes and spun at 14,000×g for 10minutes at 4° C. to remove insoluble material. The liquid phase isextracted with 200 μl of chloroform, and the upper aqueous phase isremoved to a fresh tube. The RNA is precipitated by the addition of 500μl of isopropanol and centrifuged to pellet. The aqueous phase iscarefully removed, and the pellet is washed in 75% ethanol and spun tore-collect the RNA pellet. The supernatant is carefully removed, and thepellet is air dried for 10 minutes. The RNA pellet is resuspended in 50μl of DEPC—H₂0 and analyzed by spectrophotometry at λ 260 and 280 nm todetermine yield and purity. Yields can be 1-4 mg of total RNA from 2 mlof packed worms.

Full-length cDNAs can be generated using 5′ and 3′ RACE techniques incombination with EST sequence information. The molecular technique 5′RACE (Life Technologies, Inc., Rockville, Md.) can be employed to obtaincomplete or near-complete 5′ ends of cDNA sequences for nematode ATPsynthase subunit E-like cDNA sequences. Briefly, following theinstructions provided by Life Technologies, first strand cDNA issynthesized from total nematode RNA using Murine Leukemia Virus ReverseTranscriptase (M-MLV RT) and a gene specific “antisense” primer, e.g.,designed from available EST sequence. RNase H is used to degrade theoriginal mRNA template. The first strand cDNA is separated fromunincorporated dNTPs, primers, and proteins using a GlassMAX SpinCartridge. Terminal deoxynucleotidyl transferase (TdT) is used togenerate a homopolymeric dC tailed extension by the sequential additionof dCTP nucleotides to the 3′ end of the first strand cDNA. Followingaddition of the dC homopolymeric extension, the first strand cDNA isdirectly amplified without further purification using Taq DNApolymerase, a gene specific “antisense” primer designed from availableEST sequences to anneal to a site located within the first strand cDNAmolecule, and a deoxyinosine-containing primer that anneals to thehomopolymeric dC tailed region of the cDNA in a polymerase chainreaction (PCR). 5′ RACE PCR amplification products are cloned into asuitable vector for further analysis and sequencing.

The molecular technique, 3′ RACE (Life Technologies, Inc., Rockville,Md.), can be employed to obtain complete or near-complete 3′ ends ofcDNA sequences for nematode ATP synthase subunit E-like cDNA sequences.Briefly, following the instructions provided by Life Technologies(Rockville, Md.), first strand cDNA synthesis is performed on totalnematode RNA using SuperScript™ Reverse Transcriptase and an oligo-dTprimer that anneals to the polyA tail. Following degradation of theoriginal mRNA template with RNase H, the first strand cDNA is directlyPCR amplified without further purification using Taq DNA polymerase, agene specific primer designed from available EST sequences to anneal toa site located within the first strand cDNA molecule, and a “universal”primer which contains sequence identity to 5′ end of the oligo-dTprimer. 3′ RACE PCR amplification products are cloned into a suitablevector for further analysis and sequencing.

Nucleic Acid Variants

Isolated nucleic acid molecules of the present invention include nucleicacid molecules that have an open reading frame encoding an ATP synthasesubunit E-like polypeptide. Such nucleic acid molecules includemolecules having: the sequences recited in SEQ ID NO: 1, 2, and/or 3;and sequences coding for the ATP synthase subunit E-like proteinsrecited in SEQ ID NO: 4, 5, and/or 6. These nucleic acid molecules canbe used, for example, in a hybridization assay to detect the presence ofa M. javanica, H. glycines, and/or Z. punctata nucleic acid in a sample.

The present invention includes nucleic acid molecules such as thoseshown in SEQ ID NO: 1, 2, and/or 3 that may be subjected to mutagenesisto produce single or multiple nucleotide substitutions, deletions, orinsertions. Nucleotide insertional derivatives of the nematode gene ofthe present invention include 5′ and 3′ terminal fusions as well asintra-sequence insertions of single or multiple nucleotides. Insertionalnucleotide sequence variants are those in which one or more nucleotidesare introduced into a predetermined site in the nucleotide sequence,although random insertion is also possible with suitable screening ofthe resulting product. Deletion variants are characterized by theremoval of one or more nucleotides from the sequence. Nucleotidesubstitution variants are those in which at least one nucleotide in thesequence has been removed and a different nucleotide inserted in itsplace. Such a substitution may be silent (e.g., synonymous), meaningthat the substitution does not alter the amino acid defined by thecodon. Alternatively, substitutions are designed to alter one amino acidfor another amino acid (e.g., non-synonymous). A non-synonymoussubstitution can be conservative or non-conservative. A substitution canbe such that activity, e.g., a ATP synthase subunit E-like activity, isnot impaired. A conservative amino acid substitution results in thealteration of an amino acid for a similar acting amino acid, or aminoacid of like charge, polarity, or hydrophobicity, e.g., an amino acidsubstitution listed in Table 3 below. At some positions, evenconservative amino acid substitutions can disrupt the activity of thepolypeptide.

TABLE 3 Conservative Amino Acid Replacements For Amino Code Replace withany of Alanine Ala Gly, Cys, Ser Arginine Arg Lys, His Asparagine AsnAsp, Glu, Gln, Aspartic Acid Asp Asn, Glu, Gln Cysteine Cys Met, Thr,Ser Glutamine Gln Asn, Glu, Asp Glutamic Acid Glu Asp, Asn, Gln GlycineGly Ala Histidine His Lys, Arg Isoleucine Ile Val, Leu, Met Leucine LeuVal, Ile, Met Lysine Lys Arg, His Methionine Met Ile, Leu, ValPhenylalanine Phe Tyr, His, Trp Proline Pro Serine Ser Thr, Cys, AlaThreonine Thr Ser, Met, Val Tryptophan Trp Phe, Tyr Tyrosine Tyr Phe,His Valine Val Leu, Ile, Met

The current invention also embodies splice variants of nematode ATPsynthase subunit E-like sequences.

Another aspect of the present invention embodies a polypeptide-encodingnucleic acid molecule that is capable of hybridizing under conditions oflow stringency (or high stringency) to the nucleic acid molecule putforth in SEQ ID NO: 1, 2, and/or 3, or their complements.

The nucleic acid molecules that encode for ATP synthase subunit E-likepolypeptides may correspond to the naturally occurring nucleic acidmolecules or may differ by one or more nucleotide substitutions,deletions, and/or additions. Thus, the present invention extends togenes and any functional mutants, derivatives, parts, fragments,naturally occurring polymorphisms, homologs or analogs thereof ornon-functional molecules. Such nucleic acid molecules can be used todetect polymorphisms of ATP synthase subunit E genes or ATP synthasesubunit E-like genes, e.g., in other nematodes. As mentioned below, suchmolecules are useful as genetic probes; primer sequences in theenzymatic or chemical synthesis of the gene; or in the generation ofimmunologically interactive recombinant molecules. Using the informationprovided herein, such as the nucleotide sequence SEQ ID NO: 1, 2, and/or3, a nucleic acid molecule encoding an ATP synthase subunit E-likemolecule may be obtained using standard cloning and a screeningtechniques, such as a method described herein.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, or in the form of DNA, including, for example, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. The nucleic acids may be inthe form of RNA/DNA hybrids. Single-stranded DNA or RNA can be thecoding strand, also referred to as the sense strand, or the non-codingstrand, also known as the anti-sense strand.

One embodiment of the present invention includes a recombinant nucleicacid molecule, which includes at least one isolated nucleic acidmolecule depicted in SEQ ID NO: 1, 2, and/or 3, inserted in a vectorcapable of delivering and maintaining the nucleic acid molecule into acell. The DNA molecule may be inserted into an autonomously replicatingvector (suitable vectors include, for example, pGEM3Z and pcDNA3, andderivatives thereof). The vector nucleic acid may be a bacteriophage DNAsuch as bacteriophage lambda or M13 and derivatives thereof. The vectormay be either RNA or DNA, single- or double-stranded, prokaryotic,eukaryotic, or viral. Vectors can include transposons, viral vectors,episomes, (e.g., plasmids), chromosomes inserts, and artificialchromosomes (e.g. BACs or YACs). Construction of a vector containing anucleic acid described herein can be followed by transformation of ahost cell such as a bacterium. Suitable bacterial hosts include, but arenot limited to, E. coli. Suitable eukaryotic hosts include yeast such asS. cerevisiae, other fungi, vertebrate cells, invertebrate cells (e.g.,insect cells), plant cells, human cells, human tissue cells, and wholeeukaryotic organisms. (e.g., a transgenic plant or a transgenic animal).Further, the vector nucleic acid can be used to generate a virus such asvaccinia or baculovirus.

The present invention also extends to genetic constructs designed forpolypeptide expression. Generally, the genetic construct also includes,in addition to the encoding nucleic acid molecule, elements that allowexpression, such as a promoter and regulatory sequences. The expressionvectors may contain transcriptional control sequences that controltranscriptional initiation, such as promoter, enhancer, operator, andrepressor sequences. A variety of transcriptional control sequences arewell known to those in the art and may be functional in, but are notlimited to, a bacterium, yeast, plant, or animal cell. The expressionvector can also include a translation regulatory sequence (e.g., anuntranslated 5′ sequence, an untranslated 3′ sequence, a poly A additionsite, or an internal ribosome entry site), a splicing sequence orsplicing regulatory sequence, and a transcription termination sequence.The vector can be capable of autonomous replication or it can integrateinto host DNA.

In an alternative embodiment, the DNA molecule is fused to a reportergene such as β-glucuronidase gene, β-galactosidase (lacZ),chloramphenicol-acetyltransferase gene, a gene encoding greenfluorescent protein (and variants thereof), or red fluorescent proteinfirefly luciferase gene, among others. The DNA molecule can also befused to a nucleic acid encoding a polypeptide affinity tag, e.g.glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, or the influenza HA tag. The affinity tag orreporter fusion joins the reading frames of SEQ ID NO: 1, 2, and/or 3 tothe reading frame of the reporter gene encoding the affinity tag suchthat a translational fusion is generated. Expression of the fusion generesults in translation of a single polypeptide that includes both anematode ATP synthase subunit E-like region and reporter protein oraffinity tag. The fusion can also join a fragment of the reading frameof SEQ ID NO: 1, 2, and/or 3. The fragment can encode a functionalregion of the ATP synthase subunit E-like polypeptides, a structurallyintact domain, or an epitope (e.g., a peptide of about 8, 10, 20, or 30or more amino acids). A nematode ATP synthase subunit E-like nucleicacid that includes at least one of a regulatory region (e.g., a 5′regulatory region, a promoter, an enhancer, a 5′ untranslated region, atranslational start site, a 3′ untranslated region, a polyadenylationsite, or a 3′ regulatory region) can also be fused to a heterologousnucleic acid. For example, the promoter of an ATP synthase subunitE-like nucleic acid can be fused to a heterologous nucleic acid, e.g., anucleic acid encoding a reporter protein.

Suitable cells to transform include any cell that can be transformedwith a nucleic acid molecule of the present invention. A transformedcell of the present invention is also herein referred to as arecombinant or transgenic cell. Suitable cells can either beuntransformed cells or cells that have already been transformed with atleast one nucleic acid molecule. Suitable cells for transformationaccording to the present invention can either be: (i) endogenouslycapable of expressing the ATP synthase subunit E-like protein or; (ii)capable of producing such protein after transformation with at least onenucleic acid molecule of the present invention.

In an exemplary embodiment, a nucleic acid of the invention is used togenerate a transgenic nematode strain, e.g., a transgenic C. elegansstrain. To generate such a strain, nucleic acid is injected into thegonad of a nematode, thus generating a heritable extrachromosomal arraycontaining the nucleic acid (see, e.g., Mello et al. (1991) EMBO J.10:3959-3970). The transgenic nematode can be propagated to generate astrain harboring the transgene. Nematodes of the strain can be used inscreens to identify inhibitors specific for a M. javanica, H. glycines,and/or Z. punctata ATP synthase subunit E-like gene.

Oligonucleotides

Also provided are oligonucleotides that can form stable hybrids with anucleic acid molecule of the present invention. The oligonucleotides canbe about 10 to 200 nucleotides, about 15 to 120 nucleotides, or about 17to 80 nucleotides in length, e.g., about 10, 20, 30, 40, 50, 60, 80,100, 120 nucleotides in length. The oligonucleotides can be used asprobes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit nematode ATP synthasesubunit E-like protein activity or production (e.g., antisense, triplexformation, ribozyme, and/or RNA drug-based reagents). The presentinvention includes oligonucleotides of RNA (ssRNA and dsRNA), DNA, orderivatives of either. The invention extends to the use of sucholigonucleotides to protect non-nematode organisms (for example e.g.,plants and animals) from disease by reading the viability of infectingnamatodes, e.g., using a technology described herein. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto a non-nematode organism using techniques known to those skilled inthe art, including, but not limited to, transgenic expression in plantsor animals.

Primer sequences can be used to amplify an ATP synthase subunit E-likenucleic acid or fragment thereof. For example, at least 10 cycles of PCRamplification can be used to obtain such an amplified nucleic acid.Primers can be at least about 8-40, 10-30 or 14-25 nucleotides inlength, and can anneal to a nucleic acid “template molecule”, e.g., atemplate molecule encoding an ATP synthase subunit E-like geneticsequence, or a functional part thereof, or its complementary sequence.The nucleic acid primer molecule can be any nucleotide sequence of atleast 10 nucleotides in length derived from, or contained withinsequences depicted in SEQ ID NO: 1, 2, and/or 3 and their complements.The nucleic acid template molecule may be in a recombinant form, in avirus particle, bacteriophage particle, yeast cell, animal cell, plantcell, fungal cell, or bacterial cell. A primer can be chemicallysynthesized by routine methods.

This invention embodies any ATP synthase subunit E-like sequences thatare used to identify and isolate similar genes from other organisms,including nematodes, prokaryotic organisms, and other eukaryoticorganisms, such as other animals and/or plants.

In another embodiment, the invention provides oligonucleotides that arespecific for a M. javanica, H. glycines, and/or Z. punctata ATP synthasesubunit E-like nucleic acid molecule. Such oligonucleotides can be usedin a PCR test to determine if a M. javanica, H. glycines, and/or Z.punctata nucleic acid is present in a sample, e.g., to monitor a diseasecaused M. javanica and/or H. glycines.

Protein Production

Isolated ATP synthase subunit E-like proteins from nematodes can beproduced in a number of ways, including production and recovery of therecombinant proteins and/or chemical synthesis of the protein. In oneembodiment, an isolated nematode ATP synthase subunit E-like protein isproduced by culturing a cell, e.g., a bacterial, fungal, plant, oranimal cell, capable of expressing the protein, under conditions foreffective production and recovery of the protein. The nucleic acid canbe operably linked to a heterologous promoter, e.g., an induciblepromoter or a constitutive promoter. Effective growth conditions aretypically, but not necessarily, in liquid media comprising salts, water,carbon, nitrogen, phosphate sources, minerals, and other nutrients, butmay be any solution in which ATP synthase subunit E-like proteins may beproduced.

In one embodiment, recovery of the protein may refer to collecting thegrowth solution and need not involve additional steps of purification.Proteins of the present invention, however, can be purified usingstandard purification techniques, such as, but not limited to, affinitychromatography, thermaprecipitation, immunoaffinity chromatography,ammonium sulfate precipitation, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, and others.

The ATP synthase subunit E-like polypeptide can be fused to an affinitytag, e.g., a purification handle (e.g., glutathione-S-reductase,hexa-histidine, maltose binding protein, dihydrofolate reductases, orchitin binding protein) or an epitope tag (e.g., c-myc epitope tag,FLAG™ tag, or influenza HA tag). Affinity tagged and epitope taggedproteins can be purified using routine art-known methods.

Antibodies Against ATP Synthase Subunit E-Like Polypeptides

Recombinant ATP synthase subunit E-like gene products or derivativesthereof can be used to produce immunologically interactive molecules,such as antibodies, or functional derivatives thereof. Useful antibodiesinclude those that bind to a polypeptide that has substantially the samesequence as the amino acid sequences recited in SEQ ID NO: 4, 5, and/or6, or that has at least 60% similarity over 50 or more amino acids tothese sequences. In a preferred embodiment, the antibody specificallybinds to a polypeptide having the amino acid sequence recited in SEQ IDNO: 4, 5, and/or 6. The antibodies can be antibody fragments andgenetically engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to more than one epitope. Suchantibodies may be polyclonal or monoclonal and may be selected fromnaturally occurring antibodies or may be specifically raised to arecombinant ATP synthase subunit E-like protein.

Antibodies can be derived by immunization with a recombinant or purifiedATP synthase subunit E-like gene or gene product. As used herein, theterm “antibody” refers to an immunoglobulin, or fragment thereof.Examples of antibody fragments include F(ab) and F(ab′)₂ fragments,particularly functional ones able to bind epitopes. Such fragments canbe generated by proteolytic cleavage, e.g., with pepsin, or by geneticengineering. Antibodies can be polyclonal, monoclonal, or recombinant.In addition, antibodies can be modified to be chimeric, or humanized.Further, an antibody can be coupled to a label or a toxin.

Antibodies can be generated against a full-length ATP synthase subunitE-like protein, or a fragment thereof, e.g., an antigenic peptide. Suchpolypeptides can be coupled to an adjuvant to improve immunogenicity.Polyclonal serum is produced by injection of the antigen into alaboratory animal such as a rabbit and subsequent collection of sera.Alternatively, the antigen is used to immunize mice. Lymphocytic cellsare obtained from the mice and fused with myelomas to form hybridomasproducing antibodies.

Peptides for generating ATP synthase subunit E-like antibodies can beabout 8, 10, 15, 20, 30 or more amino acid residues in length, e.g., apeptide of such length obtained from SEQ ID NO: 4, 5, and/or 6. Peptidesor epitopes can also be selected from regions exposed on the surface ofthe protein, e.g., hydrophilic or amphipathic regions. An epitope in thevicinity of the active or binding site can be selected such that anantibody binding such an epitope would block access to the active siteor prevent binding. Antibodies reactive with, or specific for, any ofthese regions, or other regions or domains described herein areprovided. An antibody to an ATP synthase subunit E-like protein canmodulate an ATP synthase subunit E-like activity.

Monoclonal antibodies, which can be produced by routine methods, areobtained in abundance and in homogenous form from hybridomas formed fromthe fusion of immortal cell lines (e.g., myelomas) with lymphocytesimmunized with ATP synthase subunit E-like polypeptides such as thoseset forth in SEQ ID NO: 4, 5, and/or 6.

In addition, antibodies can be engineered, e.g., to produce a singlechain antibody (see, for example, Colcher et al. (1999) Ann N Y Acad Sci880: 263-280; and Reiter (1996) Clin Cancer Res 2: 245-252). In stillanother implementation, antibodies are selected or modified based onscreening procedures, e.g., by screening antibodies or fragments thereoffrom a phage display library.

Antibodies of the present invention have a variety of important useswithin the scope of this invention. For example, such antibodies can beused: (i) as therapeutic compounds to passively immunize an animal inorder to protect the animal from nematodes susceptible to antibodytreatment; (ii) as reagents in experimental assays to detect presence ofnematodes; (iii) as tools to screen for expression of the gene productin nematodes, animals, fungi, bacteria, and plants; and/or (iv) as apurification tool of ATP synthase subunit E-like protein; (v) as ATPsynthase subunit E inhibitors/activators that can be expressed orintroduced into plants or animals for therapeutic purposes.

An antibody against an ATP synthase subunit E-like protein can beproduced in a plant cell, e.g., in a transgenic plant or in culture(see, e.g., U.S. Pat. No. 6,080,560).

Antibodies that specifically recognize a M. javanica, H. glycines,and/or Z. punctata ATP synthase subunit E-like proteins can be used toidentify M. javanica, H. glycines, and/or Z. punctata nematodes, and,thus, can be used to monitor a disease caused by M. javanica and/or H.glycines.

Nucleic Acids Agents

Also featured are isolated nucleic acids that are antisense to nucleicacids encoding nematode ATP synthase subunit E-like proteins. An“antisense” nucleic acid includes a sequence that is complementary tothe coding strand of a nucleic acid encoding an ATP synthase subunitE-like protein. The complementarity can be in a coding region of thecoding strand or in a noncoding region, e.g., a 5′ or 3′ untranslatedregion, e.g., the translation start site. The antisense nucleic acid canbe produced from a cellular promoter (e.g., a RNA polymerase II or IIIpromoter), or can be introduced into a cell, e.g., using a liposome. Forexample, the antisense nucleic acid can be a synthetic oligonucleotidehaving a length of 10 about 10, 15, 20, 30, 40, 50, 75, 90, 120 or morenucleotides in length.

An antisense nucleic acid can be synthesized chemically or producedusing enzymatic reagents, e.g., a ligase. An antisense nucleic acid canalso incorporate modified nucleotides, and artificial backbonestructures, e.g., phosphorothioate derivative, and acridine substitutednucleotides.

Ribozymes. The antisense nucleic acid can be a ribozyme. The ribozymecan be designed to specifically cleave RNA, e.g., an ATP synthasesubunit E-like mRNA. Methods for designing such ribozymes are describedin U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature334:585-591. For example, the ribozyme can be a derivative ofTetrahymena L-19 IVS RNA in which the nucleotide sequence of the activesite is modified to be complementary to an ATP synthase subunit E-likenucleic acid (see, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cechet al. U.S. Pat. No. 5,116,742).

Peptide Nucleic acid (PNA). An antisense agent directed against an ATPsynthase subunit E-like nucleic acid can be a peptide nucleic acid(PNA). See Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23)for methods and a description of the replacement of the deoxyribosephosphate backbone for a pseudopeptide backbone. A PNA can specificallyhybridize to DNA and RNA under conditions of low ionic strength as aresult of its electrostatic properties. The synthesis of PNA oligomerscan be performed using standard solid phase peptide synthesis protocolsas described in Hyrup et al. (1996) supra and Perry-O'Keefe et al. Proc.Natl. Acad. Sci. 93: 14670-14675.

RNA Mediated Interference (RNAi). A double stranded RNA (dsRNA) moleculecan be used to inactivate an ATP synthase subunit E-like gene in a cellby a process known as RNA mediated-interference (RNAi; e.g., Fire et al.(1998) Nature 391:806-811, and Gönczy et al. (2000) Nature 408:331-336).The dsRNA molecule can have the nucleotide sequence of an ATP synthasesubunit E-like nucleic acid described herein or a fragment thereof. Themolecule can be injected into a cell, or a syncytium, e.g., a nematodegonad as described in Fire et al., supra.

Screening Assays

Another embodiment of the present invention is a method of identifying acompound capable of altering (e.g., inhibiting or enhancing) theactivity of ATP synthase subunit E-like molecules. This method, alsoreferred to as a “screening assay,” herein, includes, but is not limitedto, the following procedure: (i) contacting an isolated ATP synthasesubunit E-like protein with a test inhibitory compound under conditionsin which, in the absence of the test compound, the protein has ATPsynthase subunit E-like activity; and (ii) determining if the testcompound alters the ATP synthase subunit E-like activity or alters theability of the subunit E to regulate other polypeptides or moleculese.g., the catalytic subcomplex of ATP synthase or a portion (subunit)thereof. Suitable inhibitors or activators that alter a nematode ATPsynthase subunit E-like activity include compounds that interactdirectly with a nematode ATP synthase subunit E-like protein, perhapsbut not necessarily, in the active or binding site. They can alsointeract with other regions of the nematode ATP synthase subunit Eprotein by binding to regions outside of the active site or siteresponsible for regulation, for example, by allosteric interaction. Theycan also bind to the complex normally bound by the subunit E,interfering with binding to and regulation by the subunit E.

Compounds. A test compound can be a large or small molecule, forexample, an organic compound with a molecular weight of about 100 to10,000; 200 to 5,000; 200 to 2000; or 200 to 1,000 daltons. A testcompound can be any chemical compound, for example, a small organicmolecule, a carbohydrate, a lipid, an amino acid, a polypeptide, anucleoside, a nucleic acid, or a peptide nucleic acid. Small moleculesinclude, but are not limited to, metabolites, metabolic analogues,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds). Compounds and componentsfor synthesis of compounds can be obtained from a commercial chemicalsupplier, e.g., Sigma-Aldrich Corp. (St. Louis, Mo.). The test compoundor compounds can be naturally occurring, synthetic, or both. A testcompound can be the only substance assayed by the method describedherein. Alternatively, a collection of test compounds can be assayedeither consecutively or concurrently by the methods described herein.

Compounds can also act by allosteric inhibition or by preventing thesubunit E from binding to, and thus, regulating its target, i.e., an ATPsynthase.

A high-throughput method can be used to screen large libraries ofchemicals. Such libraries of candidate compounds can be generated orpurchased, e.g., from Chembridge Corp. (San Diego, Calif.). Librariescan be designed to cover a diverse range of compounds. For example, alibrary can include 10,000, 50,000, or 100,000 or more unique compounds.Merely by way of illustration, a library can be constructed fromheterocycles including pyridines, indoles, quinolines, furans,pyrimidines, triazines, pyrroles, imidazoles, naphthalenes,benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines,thiphenes, thiazoles, benzothiazoles, and morpholines. A library can bedesigned and synthesized to cover such classes of chemicals, e.g., asdescribed in DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:6909-6913; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; Choet al. (1993) Science 261:1303-1305; Carrell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.

Organism-based Assays. Organisms can be grown in microtiter plates,e.g., 6-well, 32-well, 64-well, 96-well, 384-well plates.

In one embodiment, the organism is a nematode. The nematodes can begenetically modified. Non-limiting examples of such modified nematodesinclude: 1) nematodes or nematode cells (M. javanica, H. glycines, Z.punctata, and/or C. elegans) having one or more ATP synthase subunitE-like genes inactivated (e.g., using RNA mediated interference); 2)nematodes or nematode cells expressing a heterologous ATP synthasesubunit E-like gene, e.g., an ATP synthase subunit E-like gene fromanother species; and 3) nematodes or nematode cells having one or moreendogenous ATP synthase subunit E-like genes inactivated and expressinga heterologous ATP synthase subunit E-like gene, e.g., a M. javanica, H.glycines, and/or Z. punctata ATP synthase subunit E-like gene asdescribed herein.

A plurality of candidate compounds, e.g., a combinatorial library, canbe screened. The library can be provided in a format that is amenablefor robotic manipulation, e.g., in microtitre plates. Compounds can beadded to the wells of the microtiter plates. Following compound additionand incubation, viability and/or reproductive properties of thenematodes or nematode cells are monitored.

The compounds can also be pooled, and the pools tested. Positive poolsare split for subsequent analysis. Regardless of the method, compoundsthat decrease the viability or reproductive ability of nematodes,nematode cells, or progeny of the nematodes are considered leadcompounds.

In another embodiment, the compounds can be tested on a microorganism ora eukaryotic or mammalian cell line, e.g., rabbit skin cells, Chinesehamster ovary cells (CHO), and/or Hela cells. For example, CHO cellsabsent for ATP synthase subunit E-like genes, but expressing a nematodeATP synthase subunit E-like gene can be used. The generation of suchstrains is routine in the art. As described above for nematodes andnematode cells, the cell lines can be grown in microtitre plates, eachwell having a different candidate compound or pool of candidatecompounds. Growth is monitored during or after the assay to determine ifthe compound or pool of compounds is a modulator of a nematode ATPsynthase subunit E-like polypeptide.

In Vitro Activity Assays. The screening assay can be an in vitroactivity assay. For example, a nematode ATP synthase subunit E-likepolypeptide can be purified as described above. The polypeptide can bedisposed in an assay container, e.g., a well of a microtitre plate. Acandidate compound can be added to the assay container, and the ATPsynthase subunit E-like activity is measured. Optionally, the activityis compared to the activity measured in a control container in which nocandidate compound is disposed or in which an inert or non-functionalcompound is disposed

In Vitro Binding Assays. The screening assay can also be a cell-freebinding assay, e.g., an assay to identify compounds that bind a nematodeATP synthase subunit E-like polypeptide. For example, a nematode ATPsynthase subunit E-like polypeptide can be purified and labeled. Thelabeled polypeptide is contacted to beads; each bead has a tagdetectable by mass spectroscopy, and test compound, e.g., a compoundsynthesized by combinatorial chemical methods. Beads to which thelabeled polypeptide is bound are identified and analyzed by massspectroscopy. The beads can be generated using “split-and-pool”synthesis. The method can further include a second assay to determine ifthe compound alters the activity of the ATP synthase subunit E-likepolypeptide.

Optimization of a Compound. Once a lead compound has been identified,standard principles of medicinal chemistry can be used to producederivatives of the compound. Derivatives can be screened for improvedpharmacological properties, for example, efficacy, pharmacokinetics,stability, solubility, and clearance. The moieties responsible for acompound's activity in the above-described assays can be delineated byexamination of structure-activity relationships (SAR) as is commonlypracticed in the art. One can modify moieties on a lead compound andmeasure the effects of the modification on the efficacy of the compoundto thereby produce derivatives with increased potency. For an example,see Nagarajan et al. (1988) J. Antibiot. 41:1430-1438. Amodification caninclude N-acylation, amination, amidation, oxidation, reduction,alkylation, esterification, and hydroxylation. Furthermore, if thebiochemical target of the lead compound is known or determined, thestructure of the target and the lead compound can inform the design andoptimization of derivatives. Molecular modeling software to do this iscommercially available (e.g., Molecular Simulations, Inc.). “SAR byNMR,” as described in Shuker et al. (1996) Science 274:1531-1534, can beused to design ligands with increased affinity, by joininglower-affinity ligands.

A preferred compound is one that interferes with the function of an ATPsynthase subunit E-like polypeptide and that is not substantially toxicto plants, animals, or humans. By “not substantially toxic” it is meantthat the compound does not substantially affect the respective animal,or human ATP synthase subunit E proteins or ATP synthase activity. Thus,particularly desirable inhibitors of M. javanica, H. glycines, and/or Z.punctata ATP synthase subunit E do not substantially inhibit ATPsynthase subunit E-like polypeptides or ATP synthase activity ofvertebrates, e.g., humans for example. Other desirable compounds do notsubstantially inhibit to ATP synthase activity of plants.

Standard pharmaceutical procedures can be used to assess the toxicityand therapeutic efficacy of a modulator of an ATP synthase subunitE-like activity. The LD50 (the dose lethal to 50% of the population) andthe ED50 (the dose therapeutically effective in 50% of the populationcan be measured in cell cultures, experimental plants (e.g., inlaboratory or field studies), or experimental animals. Optionally, atherapeutic index can be determined which is expressed as the ratio:LD50/ED50. High therapeutic indices are indicative of a compound beingan effective ATP synthase subunit E-like inhibitor, while not causingundue toxicity or side effects to a subject (e.g., a host plant or hostanimal).

Alternatively, the ability of a candidate compound to modulate anon-nematode ATP synthase subunit E-like polypeptide is assayed, e.g.,by a method described herein. For example, the inhibition constant of acandidate compound for a mammalian ATP synthase subunit E-likepolypeptide can be measured and compared to the inhibition constant fora nematode ATP synthase subunit E-like polypeptide.

The aforementioned analyses can be used to identify and/or design amodulator with specificity for nematode ATP synthase subunit E-likepolypeptide over vertebrate or other animal (e.g., mammalian) ATPsynthase subunit E-like polypeptides. Suitable nematodes to target areany nematodes with the ATP synthase subunit E-like proteins or proteinsthat can be targeted by a compound that otherwise inhibits, reduces,activates, or generally effects the activity of nematode ATP synthasesubunit E proteins.

Inhibitors of nematode ATP synthase subunit E-like proteins can also beused to identify ATP synthase subunit E-like proteins in the nematode orother organisms using procedures known in the art, such as affinitychromatography. For example, a specific antibody may be linked to aresin and a nematode extract passed over the resin, allowing any ATPsynthase subunit E-like proteins that bind the antibody to bind theresin. Subsequent biochemical techniques familiar to those skilled inthe art can be performed to purify and identify bound ATP synthasesubunit E-like proteins.

Agricultural Compositions

A compound that is identified as an ATP synthase subunit E-likepolypeptide inhibitor can be formulated as a composition that is appliedto plants, soil, or seeds in order to confer nematode resistance. Thecomposition can be prepared in a solution, e.g., an aqueous solution, ata concentration from about 0.005% to 10%, or about 0.01% to 1%, or about0.1% to 0.5% by weight. The solution can include an organic solvent,e.g., glycerol or ethanol. The composition can be formulated with one ormore agriculturally acceptable carriers. Agricultural carriers caninclude: clay, talc, bentonite, diatomaceous earth, kaolin, silica,benzene, xylene, toluene, kerosene, N-methylpyrrolidone, alcohols(methanol, ethanol, isopropanol, n-butanol, ethylene glycol, propyleneglycol, and the like), and ketones (acetone, methylethyl ketone,cyclohexanone, and the like). The formulation can optionally furtherinclude stabilizers, spreading agents, wetting extenders, dispersingagents, sticking agents, disintegrators, and other additives, and can beprepared as a liquid, a water-soluble solid (e.g., tablet, powder orgranule), or a paste.

Prior to application, the solution can be combined with another desiredcomposition such as another antihelmintic agent, germicide, fertilizer,plant growth regulator and the like. The solution may be applied to theplant tissue, for example, by spraying, e.g., with an atomizer, bydrenching, by pasting, or by manual application, e.g., with a sponge.The solution can also be distributed from an airborne source, e.g., anaircraft or other aerial object, e.g., a fixture mounted with anapparatus for spraying the solution, the fixture being of sufficientheight to distribute the solution to the desired plant tissues.Alternatively, the composition can be applied to plant tissue from avolatile or airborne source. The source is placed in the vicinity of theplant tissue and the composition is dispersed by diffusion through theatmosphere. The source and the plant tissue to be contacted can beenclosed in an incubator, growth chamber, or greenhouse, or can be insufficient proximity that they can be outdoors.

If the composition is distributed systemically thorough the plant, thecomposition can be applied to tissues other than the leaves, e.g., tothe stems or roots. Thus, the composition can be distributed byirrigation. The composition can also be injected directly into roots orstems.

A skilled artisan would be able to determine an appropriate dosage forformulation of the active ingredient of the composition. For example,the ED50 can be determined as described above from experimental data.The data can be obtained by experimentally varying the dose of theactive ingredient to identify a dosage effective for killing a nematode,while not causing toxicity in the host plant or host animal (i.e.non-nematode animal).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated nucleic acid molecule encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:4.
 2. The isolated nucleic acidmolecule of claim 1 wherein the polypeptide consists of the amino acidsequence of SEQ ID NO:4.
 3. A vector comprising the nucleic acidmolecule of claim 1 or
 2. 4. The vector of claim 3 wherein the vector isan expression vector.
 5. An isolated recombinant cell transformed ortransfected with the isolated nucleic acid molecule of claim 1 or
 2. 6.An isolated recombinant cell transformed or transfected with the vectorof claim
 3. 7. The isolated recombinant cell of claim 6 wherein thevector is an expression vector.